1. Decoding a PNA Encoded Peptide Library by PCR: The Discovery of New Cell Surface Receptor Ligands 
Nina Svensen, Juan José Díaz-Mochón, Mark Bradley 
Chemistry & Biology 
Volume 18, Issue 10, Pages (October 2011)
DOI: /j.chembiol
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2. Chemistry & Biology 2011 18, 1284-1289DOI: (10. 1016/j. chembiol. 2011
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3. Figure 1
Screening 10,000 PNA-Encoded Peptide Ligands and PNA-Tag Amplification
(A) The general strategy for the identification of ligands for cell surface receptors by microarray analysis of a library of PNA-encoded peptide ligands.
(B) Generic structure of Library-1. AA1, AA2, and AA4 were Ile, Val, Phe, Pro, Arg, Glu, Lys, d-Pro, Ser or d-Val and AA3 Ile, Val, Phe, Ala, Pro, Arg, Glu, Lys, d-ALa, Ser, or Pro. FAM is 5(6)-carboxyfluorescein amide.
(C) The strategy of indirect amplification of the PNA-tags. The PNA extracted from cells was hybridized with a complementary ssDNA library (ssDNA-Library-1) and unhybridized ssDNA was degraded with a single-strand specific nuclease. The PNA/DNA duplexes were amplified by PCR to produce fluorescently labeled ssDNA analogous to the PNAs extracted from cells. The fluorescently labeled ssDNA was hybridized to a complementary microarray to allow decoding of the amplified ssDNA (and hence the PNA-tags and their encoded peptides).
See also Table S1.
Chemistry & Biology  , DOI: ( /j.chembiol )
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4. Figure 2 DNA Gel Electrophoresis of the Amplified PNA
(A) Proof of concept of PNA amplification. (i) oligonucleotides-2 and -3; (ii) oligonucleotide-1; (iii) dsDNA/PNA complex; (iv) oligonucleotides-1, -2 and -3 (ssDNA/dsDNA hybrid complex).
(B) Library-1: Control showing the PCR amplified 10,000 member PNA-Library-1 (ssDNA-Library-1-FAM); D54: FAM-labeled ssDNA decoded library (ssDNA-D54-FAM); CCR6: FAM-labeled ssDNA decoded library (ssDNA-HEK293T-CCR6-FAM).
See also Figure S1.
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5. Figure 3 Flow Cytometry Analysis of Peptides Binding to D54 Cells
(A) FAM versus Rho-filtered scatter plots gated for live, single cells following incubation with (v), (vii), and (viii) using a FACS Aria cytometer with FITC and PE-Texas-Red filters (10,000 populations, n = 3). D54 cells were detached by scraping for minimal destruction of the extracellular receptor moieties or by trypsin/EDTA as a control for extracellular binding of peptides.
(B) Mean FAM and Rho-filtered fluorescence of scatter plots versus incubation with the labeled peptides. Error bars indicate ± standard deviation (SD) (n = 3).
See also Figures S2 and S3.
Chemistry & Biology  , DOI: ( /j.chembiol )
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6. Figure 4
Flow Cytometry Analysis of Peptides Binding to HEK293T-CCR6 Cells
(A) FAM versus peridinin chlorophyll protein complex (PerCP)-filtered scatter plots gated for live, single cells following incubation with (vi) or anti-human CCR6 using FITC and PE-Texas-Red filters. Conditions as given in Figure 3.
(B) Mean FAM and PerCP-filtered fluorescence of scatter plots versus incubation with (vi) and the CCR6 antibodies. Error bars indicate ± SD (n = 3).
See also Figures S2 and S3.
Chemistry & Biology  , DOI: ( /j.chembiol )
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